title: Selective peptide inhibitors of bifunctional thymidylate synthase-dihydrofolate reductase from Toxoplasma gondii provide insights into domain-domain communication and allosteric regulation.
abstract: The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS-DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking β-strands at the interface, revealing a previously unknown allosteric target. The current study shows that these β-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain-domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut, 06510; Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut, 06510.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Landau
authors.forename: Hitesh
authors.initials: H
authors.lastname: Sharma
authors.forename: Karen S
authors.initials: KS
authors.lastname: Anderson
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000595:Amino Acid Sequence; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014122:Toxoplasma / Q000201:enzymology*
chemical_list: D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D010455:Peptides; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase
keywords: allosteric sites; conformational changes; enzyme kinetics; peptide inhibition; species-selectivity